Does CRISPR-Cas immunity limit the effectiveness of phage therapy?

Globally rising rates of resistance against all major classes of chemotherapeutic antibiotics has renewed interest in the therapeutic use of phages to treat bacterial infections. As with antibiotic treatments, the emergence of bacterial resistance against phage treatments could also limit the long-term sustainability of phage-based therapeutics. We currently lack a mechanistic understanding of how bacteria evolve resistance against combinations of multiple phages, limiting our ability to design phage combinations that limit resistance. Importantly, bacteria possess multiple distinct mechanisms to defend against phage infection, including surface receptor modification (SRM) and CRISPR-Cas. These distinct mechanisms are favoured under different conditions which are likely to vary at the site of infection, but the importance of CRISPR-Cas for the evolution of resistance against phage therapy is unknown. We will use laboratory evolution experiments to determine how CRISPR-Cas immunity affects the evolution of multi-phage resistance across infection-relevant environmental conditions predicted to tip the balance from SRM to CRISPR-Cas immunity. We will then test if, by enhancing multi-phage resistance evolution, CRISPR-Cas immunity reduces the efficacy of phage therapy in vivo using a mouse model of acute respiratory infection. These experiments will develop and test an evolutionary framework for understanding how CRISPR-Cas affects multi-phage resistance evolution and the efficacy of phage therapy.

Phage therapy is a promising alternative to antibiotics for treating some bacterial infections. It involves the application of combinations of phages, but like antibiotics the effectiveness of phage therapy can be limited by the evolution of resistance. At present we lack a mechanistic understanding of the evolution of multi-phage resistance in bacteria. In particular, the role of CRISPR-Cas, which by providing an additional modality of resistance is likely to promote multi-phage resistance, is unknown. Here we will combine laboratory evolution experiments and experimental infections in mice to test how the efficacy of phage therapy against Pseudomonas aeruginosa PA14 is affected by whether the bacteria do or do not encode a CRISPR-Cas locus. First, we use a high-throughput experimental evolution to test the effect of CRISPR-Cas on the evolution of multi-phage resistance against all pairwise combinations (n=378) of 28 genetically diverse phages. Second, for a representative subset of phages we expand this experimental evolution approach to test the effect of CRISPR-Cas on multi-phage resistance evolution across a wide range of environmental conditions likely to vary at the site of infection and predicted to tip the balance from mutation-based to CRISPR-Cas resistance. Third, we test how CRISPR-Cas affects the efficacy of therapeutic phage combinations against P. aeruginosa in an established mouse model of respiratory infection where inhalation-based phage therapy has been shown to be effective at controlling P. aeruginosa strains lacking CRISPR-Cas.

Who will benefit from this research and how?

Phage therapy has a potentially important role to play in combatting globally rising rates antimicrobial resistance and has been used successfully to treat multidrug resistant infections both in the UK and overseas. Our proposal will address a major knowledge gap in terms of understanding the mechanisms of the evolution of resistance against phage combinations, as are typically used in phage therapy. Nonetheless, much work is required by industry, in terms of developing effective phage therapies that minimise resistance evolution, policy makers, in terms of removing the regulatory and economic barriers to commercialising phage therapy, and the general public, in terms of educating communities about phages and why they may be a useful alternative to traditional chemotherapeutic antibiotics in the clinic and to prevent animal and plant bacterial disease more broadly. The key benefits deriving from this research will therefore be increased knowledge and understanding of resistance evolution against phage therapy among the following groups:

1. Industrial stakeholders: Numerous small- and medium-sized enterprises and spin-out companies are developing phage-based therapeutics (e.g. we have on-going contacts with Evolution Biotechnologies and APS Biocontrol) but often without the necessary knowledge base to minimise the emergence of resistance against their chosen phage combinations. This research will provide the required evidence to define the evolutionary principles for the design of more effective phage combinations, and their optimal deployment based upon the CRISPR-status of the targeted infecting organism. Although our work will not lead directly to commercial phage combinations it will provide a general evolutionary framework that can be used in development of therapeutic phage combinations.

2. Policy makers and practitioners in healthcare and agri-food sectors: Bacterial disease and increasing rates of antimicrobial resistance are major challenges for human and animal health and necessitate that alternative treatments be investigated. Moreover, the need to restrict uses of antibiotics that remain effective mean that alternatives are required to prevent unnecessary use of antibiotics e.g. in veterinary and plant health applications. Phage therapy is a promising alternative and the subject of on-going clinical trials and successful medical case reports. Understanding of resistance evolution in phage therapy could help prevent a similar resistance crisis as that affecting antibiotics by allowing the design of treatments that minimise resistance selection; our findings will provide this evidence base and will be communicated to healthcare and agri-food stakeholders.

3. Secondary school age children: Teaching of evolution in Key Stages 2 and 3 of the National Curriculum is mainly theoretical and lacking in engaging practical classes. Our discoveries will advance understanding of the fundamental processes of bacterial evolution. Thus, we aim to add knowledge via curriculum changes and to change schoolchildren perceptions and knowledge (via our Pathways to Impact strategy). We will take experimental evolution into the school classroom allowing pupils to experience evolution in action themselves in real time, generating excitement about microbes and evolution and offering deeper experiential learning.

4. General public: Bacterial evolution is high on the news agenda due to the crisis in antimicrobial resistance (AMR), however few non-scientists realise that phage therapy is a possible alternative to antibiotics. Public engagement activities will enhance public understanding of phage therapy and put this into the context of AMR to show what we can all do to reduce the risks of AMR and explain why alternative strategies like phage therapy may be required to tackle the crisis.